Hi everyone,
I am conducting an experiment to detect copy number variation among my gDNA samples using RT-PCR. I have generated standard curve for the genes of my interests and two controls (genes known to be single copied) using synthetic DNA. When I was testing my gDNA samples, the two controls (A and B) showed very different results - thousands of fold difference after being converted into copy numbers. Among my genes of interest, 1 out of 7 genes showed the similar copy numbers to control A, and the other 6 genes showed similar copy numbers to control B.
I was wondering if anyone had similar experience and why this would happen?
Thanks for your time and help!
I have done different experiments to test the copies of Tnt1 elements in transgenic soybean. There are some comments for you:
- Make sure you have a good control gene that have a stable copy number in your tested samples (it must be an endogenous gene)
- DNA concentration for qPCR need to be accurate. I use 1ng/microlite concentration and use 1 microlite for qPCR
- It not very accurate if the copies of genes are so close (1 and 2 copies)
- Use good method to compare (I used 2^-Delta Delta Ct )
Goof luck!
What are your primer efficiencies?
And double check your calculations.
you have to do some initial calculation and experiment with acculrate dilutions. You can also check melt curve analysis and primer as well as PCR efficiency.
All the best.
- Badges
- Science method